The present invention provides an assay system for determining therapeutic activity for treating restenosis, atherosclerosis, chronic rejection syndrome and graft versus host disease (GVHD) by measuring inhibition of cell migration activity in smooth muscle cells expressing a US28 receptor from the CMV genome. Specifically, the present invention provides a method for measuring inhibition of cell migration in isolated cells transfected with US28 or infected with CMV and stimulated with a ligand. The invention further provides a method for treating restenosis, atherosclerosis, chronic rejection syndrome and GVHD by administering KHSV encoded vMIP-2, fractalkine or herbimycin.
Atherosclerosis, Restenosis, Chronic Rejection Syndrome and GVHD
Atherosclerosis is a major cause of morbidity in the industrialized world. Atherosclerotic lesions usually become apparent in adult patients as a result of complete occlusion of a strategic blood vessel and the resulting complication. However, such lesions begin much earlier in the life of the patient. It was later noticed that there was a statistical association with viral infection, particularly CMV.
It has been postulated that CMV and possibly herpes virus are involved in the inducement of atherosclerotic lesions. Several investigators have demonstrated the presence of CMV nucleic acids and/or antigens in the human arterial wall using DNA hybridization techniques (Melnick et al., Lancet 11:644-647, 1983), immunohistochemistry (Petrie et al., J. Infect. Dis. 155:158-159, 1987), dot blot and in situ hybridization techniques (Hendrix et al., Am. J. Path. 134:1151-1157, 1989), and by polymerase chain reaction (PCR) techniques using probes derived from immediate early and late genomic regions (Hendrix et al., Am. J. Path. 136:23-28, 1990). Thus, there has been finding of viral antigens and nucleic acid sequences in arterial smooth muscle cells that suggest that CMV infection of the arterial wall may be a common occurrence in patients with atherosclerosis.
Soon after renal transplantation became an accepted treatment, an association was noted between CMV infection, glomerulopathy, and rejection of the transplanted kidney. Thus, CMV was investigated to determine if it played a role in graft atherosclerosis that frequently occurs after heart transplantation (Grattan et al., J. Am. Med. Assn. 261:3561-3566, 1989). The findings show that heart transplant patients who are immunosuppressed and become infected with CMV are particularly prone to develop atherosclerosis in the transplanted organ. It is postulated that the artery wall may be the site of CMV latency because CMV DNA but not infectious virus was found in the artery wall.
Role of Chemokines
Chemokines are chemoattratants for neutrophils, monocytes, lymphocytes and bone marrow progenitors, as well as other cell types. The family of chemokines comprises four subfamilies, defined by the distribution of cysteine residues in the N terminus of these factors, the CXC, CC, C, and CX3C subfamilies. The chemokines are related by primary structure, particularly by conservation of a four-cysteine motif. C-C chemokines include such members as human monocyte chemotactic protein 1 (MCP-1), RANTES, and the macrophage inflammatory proteins 1xcex1 and 1xcex2 (MIP-1xcex1 and MIP-1xcex2). These ligands exhibit chemoattractant potential for monocytes but not neutrophils. CMV infection can also modify the level of chemokines. The level of RANTES (a chemokine) produced by cells recovered by bronchoalveolar lavage from lung transplant patients with CMV pneumonitis shows that cells from infected patients secreted greater amounts of RANTES than did cells recovered from either patients undergoing acute rejection or from control subjects (Monti et al., Transplantation 61:1757-1762, 1996). AIDS patients with CMV encephlitis have higher concentrations of MCP-1 (a chemokine) but not other chemokines in their spinal fluid than do HIV seropositive persons who are asymptomatic or AIDS patients with a number of other opportunistic infections of the central nervous system (Bernasconi et al., J. Infec. Dis. 174:1098-1101, 1996). When fibroblasts were infected with CMV, RANTES mRNA and protein expression are induced early, but extracellular RANTES accumulation, but not transcription is down-regulated late during CMV infection (Michelson et al., J. Virol. 71:6495-6500, 1997). Therefore, CMV infection has the capacity to both induce cell migration and enhance chemokine production early during the infection process.
Chemokine receptors tend to be multiple membrane-spanning proteins, generally 7 or 8 membrane-spanning proteins and tend to transduce signal through G-coupled protein signal transduction. Human C-C chemokines tend to bind to the US28 receptor of CMV (Neote et al., Cell 72:415-425, 1993). There is also a sequence homology between the C-CKR-1 receptor (normal human gene) and the CMV US28 sequence in the open reading frame region. (Neote et al., 1993). Thus, Neote et al. speculated that xe2x80x9cthe protein encoded by the US28 open reading frame of Towne strain CMV can bind C-C chemokines but not the C-X-C chemokine IL-8. However, none of the earlier chemokine receptor papers, including Neote et al., has made the connection between US28 and it s role in mediating smooth muscle cell proliferation.
The present invention provides an assay for determining therapeutic activity of US28 receptor antagonists, comprising (a) obtaining and isolating smooth muscle cells into a first chamber of a migration device, wherein the first migration chamber comprises growth media chambers and is defined by a first side of a membrane and chamber walls, and wherein the migration device comprises a second chamber defined by the second side of the membrane and having an enclosed space; (b) infecting the smooth muscle cells with human cytomegalovirus (HCVM) containing a gene encoding the US28 receptor; (c) adding a candidate therapeutic agent to the first chamber; and (d) determining the amount of cellular migration into the second chamber, whereby inhibition of cellular migration of infected smooth muscle cells indicates therapeutic activity. Preferably, the smooth muscle cells are isolated from pulmonary arteries. Preferably, the membrane has a pore size of from about 2 to about 10 microns. Most preferably, the membrane pore size is about 3 microns. Preferably the amount of cellular migration is determined by an assay for counting the number of smooth muscle cells in the second chamber wherein the assay for counting the number of smooth muscle cells is selected from the group consisting of microscopic cell counting per unit area, radiolabeling the smooth muscle cells and counting radioactivity in the second chamber, attaching a fluorescent probe to the smooth muscle cells and measuring fluorescence within the second chamber, and combinations thereof.
The present invention further provides a method for treating atherosclerosis, restenosis, chronic rejection syndrome and graft versus host disease (GVHD), comprising administering an effective amount of an agent that is a US28 receptor antagonist, wherein a US28 receptor antagonist comprises an inhibitor compound that prevents transduction of US28 receptor signal stimulated by a US28 receptor ligand, wherein a US28 receptor ligand is selected from the group consisting of RANTES, MIP-1xcex1 and MCP. Preferably, the US28 receptor antagonist is selected from the group consisting of an antibody that binds to an extracellular portion of the US28 receptor, and an antisense oligonucleotide having a nucleic acid sequence antisense to the US28 cDNA and inhibiting translation of US28 expression in infected smooth muscle cells, or a US28 binding antagonist, wherein the US28 binding antagonist is selected from the group consisting of KHSV encoded vMIP-2, fractalkine, and herbimycin. Preferably, the monoclonal antibody is chimeric or humanized by means for humanizing non-human antibodies. Preferably, the US28 antisense sequences are selected from the group consisting of SEQ ID NOS. 2-28.
The present invention further provides a method for enhancing cellular migration, comprising infecting a cell with a viral nucleic acid containing a gene encoding CVM US28 receptor or tansfecting a cell with a vector comprising the cDNA sequence for US28 operably linked to a viral promoter sequence, and stimulating the transfected or infected cell with a US28 receptor ligand, selected from the group consisting of RANTES, MIP-1xcex1 and MCP1.